Procaine salts of biosynthetic penicillins



Patented Oct. 31, 1 950 I PROCAINE SALTS OF BIOSYNTHETIC PENICILLINS Harley W. Rhodehamel, Jr., Indianapolis, Ind., assignor to Eli Lilly and Company, Indianapolis, Ind., a corporation of Indiana No Drawing. Application 'JulyI, 1948, Serial No. 36,457

1 40mins. (01.260-302) I 2 My invention relates to salts of biosynthetic tion to yield the insoluble procaine salt. The inpenicillins and more particularly to insoluble salts soluble salt which precipitates is isolated, as by of halobenzyl penicillins. filtration, and freed from traces of solvent in any An object of my invention is to prepare insolsuitable manner, such as subjecting the salt to a uble salts of halobenzyl penicillins. Another ob- 5 vacuum.

ject is to provide insoluble salts useful in the isola- It is desirable to employ an amount of procaine tion and purification of blosynthetic pencillins. salt or procaine base in slight stoichiometric ex- Another object is to provide insoluble salts of halocess to assure the complete reaction of all of the benzyl penicillins which have a prolonged therabiosynthetic penicillin present. The procaine peutic eflzect. Other objects will be apparent l0 saltsof this invention being substantially insolfrom the following disclosure of my invention. uble in water and the common organic solvents.

I have discovered that halobenzyl pe i illins precipitate when prepared in one of these solvents, form crystalline salts with procaine-and that such leaving the excess procaine or soluble salt thereof salts possess properties whereby the abov d in solution. It is believed that the procaine salt other object are accomplished, 16 of the biosynthetic penicillin comprises the com- The novel salts of my invention may be reprebination of the acidic carboxyl roup of the penisented by the following formula COOH wherein X represents a monohalogen substituent cillin with the basicnitrogen of the diethylaminohaving an atomic number less than 53. Y ethyl group in the procaine molecule.

Illustrative examples of compounds within the The biosynthetic penicillin may be recovered scope of my invention are the procaine salts of from its combination with procaine by dissolving o-fiuorobenzyl penicillin, and m-chlorobenzyl or suspending the procaine salt in aqueous solupenicillin. Procedures for the preparation of the tion, acidifying the solution to about pH 2, and biosynthetic penicillins are described in detail in extracting the biosynthetic penicillin with a penithe publication by Behrens et al., J. Biol. Chem., cillin solvent such as amyl acetate, chloroform 175, 793 (1948). and the like. From the solution thus obtained The above-mentioned procaine salts of halothe biosynthetic penicillin may be recovered by benzyl penicillinsmay be prepared by chemical any of the several suitable known processes.

reactions of addition or metathesis. The acidic 'The following examples describe specific prep:

form of the biosynthetic penicillin may be reacted arations of several salts of my invention.

with procaine base, or a soluble metallic or metal- Example 1 line (e. g. ammonium) salt of the biosynthetic penicillin may be reacted with a soluble acid addi- The procaine salt of o-fiuorobenzyl penicillin tion salt of procaine. Thus, for example, the represented by the formula OOH acidic form of the biosynthetic penicillin and is obtained as follows:

procaine base are mixed together in amyl acetate o-Fluorobenzyl penicillin used in preparing the to yield as a precipitate, the desired procaine salt above salt may be produced by growing a penicilof the biosynthetic penicillin. Alternatively, the lip-producing mold in a culture medium consodium salt of the biosynthetic penicillin and protaining about 0.02 percent of N-(2-hydroxy- 'caine hydrochloride are reacted in aqueous soluethyl) -o-fluoroacetamide, and the biosynthetic 3 penicillin produced in the broth may be isolated as the sodium salt.

To about 5.2 parts of the sodium salt of the o-fluorobenzyl penicillin dissolved in about 80 parts of water is added a solution of about 5 parts percent excess) of procaine hydrochloride in. 15 parts of water. An immediate crystalline precipitate of the procaine salt of o-fiuorobenzyl penicillin separates. The mixture is cooled to about 0 C. for about 2 hours and is then filtered to separate the insoluble procaine salt of o-fiuorobenzyl penicillin. The separated salt is washed with a small amount of cold water and is then dried in vacuo.

Example 2 The procaine salt of m-fluorobenzyl pencillin represented by the following formula procaine salt are suspended in about 50 ml. 0! cold water and suillcient dilute sulfuric acid is added slowly to acidity thesolution to about pH 2. The acidified mixture isextracted with about 50 ml. of amyl acetate whereupon the D-bromobenzyl penicillin dissolves in the amyl acetate, and the procaine in the form of its sulfate salt dissolves in the aqueous phase. The amyl acetate layer is separated from the aqueous layer and the p-bromobenzyl penicillin is recovered in purified form from the amyl acetate by extraction of the amyl acetate with cold dilute aqueous alkali.

By proceeding through the insoluble procaine penicillin salt, substantial purification oi the pbromobenzyl penicillin is eilected, the degree of purification amounting to percent or more. If a more greatly purified p-bromobenzyl penicil OOH may be prepared as follows:

m-Fluorobenzyi penicillin is obtained by growing a penicillin-producing mold in a culture medium containing about 0.02 percent of N-(2- hydroxyethyl) -m-fiuoroacetamide, and is isolated as its sodium salt. The procaine salt of m-fluorobenzyl penicillin is obtained from equal proportions of the sodium salt of m-fiuorobenzyl penicillin and procaine hydrochloride by the procedur described in Example 1.

Example 3 Instead of using a soluble salt of the penicillin and a procaine salt as described in Examples 1 and 2, the reaction of the acid form of the penicillin and the procaine base may be used. The biosynthetic penicillin in acid form is obtained by extraction of the acidified broth.

The procaine salt of p-bromobenzyl penicillin represented by the following formula 3 tool;

may be prepared as follows: p-bromobenzyl penicillin is obtained by growing a penicillin-producing mold in a culture medium containing about 0.02 percent of N-(2-hydroxyethyl), -pbromoacetamide, and extracting the culture medium or broth with amyl acetate. The amyl acetate is clarified with about 0.5 percent of decolorizing carbon or fullers earth and is concentrated by evaporation in vacuo. To about 4 liters of this amyl acetate solution containing about 3 g. of pbromobenzyl penicillin in acidic form are added slowly and with stirring 109 cc. of amyl acetate containing about 2.5 g. of procaine base. The procaine salt of p-bromobenzyl penicillin precipitates in crystalline form. The solution is cooled to about 0 C. for about 2 hours and is then filtered to isolate the precipitated procaine salt of p-br omobenzyl penicillin. The isolated crystalline salt is washed with a small amount of cold amyl acetate and dried in vacuo to remove traces of moisture and residual amyl acetate.

The p-bromobenzyl penicillin is recovered from lin is desired, the procaine salt oi the penicillin is recrystallized from alcohol or other suitable solvent prior to the recovery oi. the biosynthetic penicillin therefrom. Recovery of the p-bromobenzyl penicillin from the purified procaine salt yields the biosynthetic penicillin in substantially pure form.

For therapeutic purposes, the procaine salts of the biosynthetic penicillins or this invention preterably are administered intramuscularly as finely divided suspensions of the salts in liquid vehicles. Suitable vehicles for the provision of lnjectable suspensions include aqueous and oleaginous vehicles, for example, water, and vegetable oils such as cottonseed oil, sesame oil, and the like. As will be readily understood, the particle size of the salt in suspension should be sufiiciently small to permit the particles to pass easily through the lumen of a hypodermic needle without packing within and blocking the needle. Illustratively, a particle size which avoids blocking of a 20-gauge needle is that which will pass through a 200- mesh screen.

By way of illustration, an aqueous medicinal composition which upon intramuscular injection is capable of maintaining a therapeutically effective biosynthetic penicillin blood level for a period of about 24 hours comprises a suspension containing about 300 mg. of finely divided procaine salt of p-bromobenzyl penicillin per ml. of water.

Since, as is known, penicillins are relatively unstable in the presence of appreciable amounts of moisture, aqueous compositions containing the novel procaine salts of this invention should be used within a comparatively short time after their preparation. For this reason it is often desirable to provide the procaine salts in oleaginous'vehlcles. A suitable oleaglnous composition which will retain therapeutic potency over a long period of time is prepared by suspending in about 5 ml. of sesame oil 2 g. of the procaine salt 01' p-bromoits insoluble procaine salt as follows: 2 g. of the benzyl penicillin, ground to a sufllciently small 5 6 particle size so that all of the material will pass I claim as my invention: through a 200-mesh screen. Intramuscular inl. The procaine salt of a, halobenzyl penicillin jection of 1 ml. of this suspension will maintain represented by the formula 001-1 a therapeutically efiective blood concentration of in which X represents a monohalogen substitp-bromobenzyl penicillin for at least 24 hours. uent having an atomic number less than 53.

It i t be understood that th abov -men- 2. The procaine salt of o-fluorobenzyl penicillin tioned therapeutic compositions are given by way represented by the formula of illustration only and it will be apparent to 3. The procaine salt of m-fluorobenzyl penicilthose skilled in the art that numerous modiflcalin represented by the formula F o s @ClEh-PJ-NH-CH-C owmh v J1 I-eri.mN--coowmnmmm):

A, 60011 tions and variations of the above medicinal com- 4. The procaine salt of p-bromobenzyl penicilpositions may be made. lin represented by the formula REFERENCES CITED The following references are of record in the 40 file of this patent:

Drug and Cosmetic Industry, November 1947, page 595. v 

1. THE PROCAINE SALT OF A HALOBENZYL PENICILLIN REPRESENTED BY THE FORMULA 